RESEARCH ARTICLE
ADAMTS-1 in abdominal aortic aneurysm Emina Vorkapic1, Maggie Folkesson1†, Kerstin Magnell2, Mohammad Bohlooly-Y2, Toste La¨nne3,4, Dick Wågsa¨ter1* 1 Division of Drug Research, Department of Medical and Health Sciences, Linko¨ping University, Linko¨ping, Sweden, 2 Division of Discovery Sciences, Innovative Medicines, AstraZeneca R&D, Mo¨lndal, Sweden, 3 Division of Cardiovascular Medicine, Department of Medical and Health Sciences, Faculty of Health Sciences, Linko¨ping University, Linko¨ping, Sweden, 4 Department of Cardiovascular Surgery, County ¨ stergo¨tland, Linko¨ping, Sweden Council of O † Deceased. * [email protected]
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Abstract Introduction
OPEN ACCESS Citation: Vorkapic E, Folkesson M, Magnell K, Bohlooly-Y M, La¨nne T, Wågsa¨ter D (2017) ADAMTS-1 in abdominal aortic aneurysm. PLoS ONE 12(6): e0178729. https://doi.org/10.1371/ journal.pone.0178729 Editor: Michael Bader, Max Delbruck Centrum fur Molekulare Medizin Berlin Buch, GERMANY Received: February 1, 2017 Accepted: May 17, 2017 Published: June 1, 2017 Copyright: © 2017 Vorkapic et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Swedish Research Council (vr.se, Grant #2016-02626, DW), the Swedish Heart-Lung Foundation (hjartlungfonden.se, Grant#20160779, DW and #20130650, TL) and Åke Wibergs stiftelse (ake.wiberg.se, Grant#M16-0070, DW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Extracellular matrix degradation is a hallmark of abdominal aortic aneurysm (AAA). Among proteases that are capable of degrading extracellular matrix are a disintegrin and metalloproteases with thrombospondin motifs (ADAMTS). Pathogenesis of these proteases in AAA has not been investigated until date.
Methods and results Human aneurysmal and control aortas were collected and analyzed with RT-PCR measuring the ADAMTS-1, 4,5,6,8,9,10,13,17 and ADAMTSL-1. Expression of a majority of the investigated ADAMTS members on mRNA level was decreased in aneurysm compared to control aorta. ADAMTS-1 was one of the members that was reduced most. Protein analysis using immunohistochemistry and western blot for localization and expression of ADAMTS-1 revealed that ADAMTS-1 was present predominantly in areas of SMCs and macrophages in aneurysmal aorta and higher expressed in AAA compared to control aortas. The role of ADAMTS-1 in AAA disease was further examined using ADAMTS-1 transgenic/apoE-/mice with the experimental angiotensin II induced aneurysmal model. Transgenic mice overexpressing ADAMTS-1 showed to be similar to ADAMTS-1 wild type mice pertaining collagen, elastin content and aortic diameter.
Conclusion Several of the ADAMTS members, and especially ADAMTS-1, are down regulated at mRNA level in AAA, due to unknown mechanisms, at the same time ADAMTS-1 protein is induced. The cleavage of its substrates, don’t seem to be crucial for the pathogenesis of AAA but rather more important in the development of thoracic aortic aneurysm and atherosclerosis as shown in previous studies.
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ADAMTS-1 in abdominal aortic aneurysm
Competing interests: AstraZeneca R&D provided support in the form of salaries for authors KM and MB-Y, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
Introduction Numerous groups of proteases have been suggested to be involved in the remodelling of the extracellular matrix. The most studied family of matrix-degrading enzymes, which we also contributed to, is probably the matrix metalloproteinases (MMPs) since these proteases are capable to degrade plenty of macromolecules present in the connective tissue matrix [1–3]. The extensive family of matrix metalloproteases beside MMPs includes a disintegrin and metalloproteinase (ADAMs) of which several members have been found in tissue from abdominal aortic aneurysms (AAA) [4, 5]. Closely related to the ADAM family are a disintegrin and metalloproteinase with thrombospondin motifs, (ADAMTS). ADAMTSs are secreted into the extracellular domain and some members of this family, in contrast to other metalloproteases, have capacity to bind to extracellular matrix [6]. ADAMTS similar to other metalloproteases contain a pro-domain that preserves ADAMTSs latency. Not all the substrates for ADAMTSs are known but they usually have various matrix proteins as substrate [7]. ADAMTS-1 is highly expressed in atherosclerotic aorta and promotes atherogenesis by cleaving extracellular matrix proteins such as versican and inducing vascular smooth muscle cell migration [8]. We previously found that ADAMTS-4 and -8 are inflammatory regulated enzymes expressed in LDLR-/-/apoB100/100 mice and in macrophage-rich areas of human atherosclerotic carotid plaques and coronary unstable plaques [9]. AAA occurs when extracellular matrix of the aorta has degenerated, and the aortic diameter enlarges irreversibly with a deadly outcome in the form of rupture. None of the currently known members of ADAMTS family have been studied in relation to AAA. In this study we focused our investigation on the presence and expression of ADAMTS-1. We also included mRNA expression analysis of several other members of our interest such as 4, 5, 6, 8, 9, 10, 13, 17 and ADAMTSL-1 in the aortic wall of control aortas and AAA. The role of ADAMTS-1 in AAA was further investigated using angiotensin II induced AAA in mice. Our data evaluating the expression of ADAMTS members in human aneurysm shows that mRNA expression of several of the members are decreased in AAA samples as compared to control aortas. Mice overexpressing ADAMTS-1 in an angiotensin II induced AAA develop aneurysm to the same extent as wild type mice and therefore ADAMTS-1 probably plays minor role in AAA progression.
Materials and methods Human samples AAA patients (n = 16, all men with mean age of 66, range 56–74 with aortic diameter of 65 mm, range 47–75) undergoing surgical repair were included in this study and biopsies from thrombus-free (NTH, n = 12) and thrombus-covered (TH, n = 16) aneurysm wall were taken since previous studies have revealed major differences in inflammatory response between aneurysm walls covered and not covered by an intraluminal thrombus [2]. Samples included were sufficient to detect a difference of two-fold with 95% confidence interval and at >80% power. Aortic walls were assayed as whole aorta and part of them were dissected to adventitia and media in aneurysmal aorta. Aorta from organ donors (n = 13, all men with mean age of 49, range 32–61) without clinical or macroscopic signs of aortic atherosclerosis or aneurysm were used as non-aneurysmal external controls. Biopsies were immediately fixed in 4% formaldehyde for histology analyses, snap frozen in liquid nitrogen for protein analysis or placed in RNAlater (Ambion, Austin, TX, USA) overnight in 40 C and then stored in -800 C for RNA isolation.
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ADAMTS-1 in abdominal aortic aneurysm
All AAA participants gave written informed consent to the study, which was approved by the regional ethical review board in Linko¨ping, Sweden. The use of organ donors was approved by the regional ethical review board in Lund, Sweden.
AngII-induced AAA in mice AngII-induced aneurysm is an inflammation-driven model that is frequently used to experimentally induce AAA [10–12]. Aneurysm was induced in wild type male hypercholesterolemic apoE-/- mice (Taconic, Bomholt, Denmark) mice (n = 12) and apoE-/-/ADAMTS-1 transgenic mice (n = 14), established and obtained from Astrazeneca, Gothenburg, Sweden as previously described [8]. Animals included were sufficient to detect a 20% difference with 95% confidence interval at >80% power. Animals were housed in groups in pathogen free (SPF) facility in IVC enriched cages and maintained on a 12:12 hour light/dark cycle. At eight weeks of age, AAA was induced, in randomly chosen mice, by chronic infusion of 1000 ng/kg/min AngII (Cat.no.9525, Sigma Aldrich, St. Louis, USA) via mini-osmotic pumps (Model 1004, Alzet, CA, USA) as described previously [11]. Pumps were placed under anesthesia using isoflurane. Post-operative pain was treated with buprenorphine at a dose of 0.1 mg/ kg body weight, injected subcutaneously twice per day for 3 days. A group of apoE-/- mice were infused with 0.9% NaCl and were used as control mice. Standard Chow diet and water was allowed ad libidum throughout the whole study and mice were monitored daily for signs of discomfort. After 28 days, mice were sacrificed. The aorta was removed and fixated in RNAlater for 24 hours thereafter frozen in -70˚C for gene expression analysis or snap frozen for protein analysis. Part of aorta was fixed in formaldehyde and embedded in paraffin for histology and analysis of aneurysm formation. The study was approved by the local ethical committee in Linko¨ping, Sweden (47–13).
Immunohistochemistry Paraffin-embedded human abdominal aortas from four patients were sectioned (5 μm) and rehydrated in several changes of ethanol and Tissue-Clear1 (Sakura Finetek, Leiden, The Netherlands). Endogenous peroxidase activity was quenched by treatment with 3% hydrogen peroxide for 5 min followed by incubation in 5% blocking rabbit or goat serum albumin solution. Sections were then incubated with human primary antibodies against ADAMTS-1 (2 μg/ ml, Cat. No. AF5867, R&D Systems, Inc, Minneapolis, USA), CD68 (0.1 μg/ml, Cat. No. NCL-CD68-KP1, Leica Microsystems, Newcastle, UK) and α-actin (0.6 mg/ml, cat. No. A5228, clone 1A4, Sigma-Aldrich) at 4˚C overnight followed by secondary biotinylated rabbit anti-sheep IgG or goat anti-mouse IgG antibody (Dako, Stockholm, Sweden). Avidin-biotin peroxidase complexes (Dako) were added followed by visualization with 3,30 -diaminobenzidine tetrahydrochloride (Dako). All sections were counterstained with Mayer’s hematoxylin (Histolab Products, Go¨teborg, Sweden).
Western blot Frozen aortas from AAA patients and non-aneurysmal control donors were homogenised in ice-cold RIPA lysis buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% Na-deoxy-cholate, 1% Triton-100 and 0.1% SDS, 100 μg/ml phenylmethylsulphonyl fluoride (PMSF) and protease inhibitor cocktail (Roche Diagnostics Scandinavia, Stockholm, Sweden). Protein quantification of tissue extracts (30 μg of protein) were separated under reducing conditions using 4–12% gradient SDS-PAGE and transferred onto a polyvinylidine difluoride membrane (Amersham Life Science, Amersham, UK). Membranes were blocked in 5% non-fat dry milk and incubation with primary rabbit ADAMTS-1 antibody (1 μg/ml, Cat.
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ADAMTS-1 in abdominal aortic aneurysm
No. AF5867, R&D Systems) overnight at 4˚C followed by incubation by a horseradish peroxidase-conjugated secondary anti-sheep antibody (1:5000, Cat. No. sc-2770, Santa Cruz Biotechnology, Heidelberg, Germany). Immunoreactive proteins were visualized by chemiluminescence ECL Prime Western blotting detection system (Amersham). Loading was controlled using coomassie blue staining of membranes.
Elastin and collagen staining Paraffin-embedded sections (5 μm) from mice aorta were rehydrated in several changes of ethanol and Tissue-Clear1 (Sakura Finetek). Sections were then stained for collagen and elastin as previously described [13]. Sections were studied under light microscopy ZEN 2012 (Zeiss, Jena, Germany). Quantification of collagen and elastin were performed blinded. A scoring system from 1 to 4 was used with 1 defined as no degradation of elastin, 2 as low degradation, 3 as intermediate degradation, and 4 as high degradation. For collagen quantification, 1 defined as low amount of collagen in all layers of the aorta, 2 as low amount of collagen in aorta media or in aorta adventitia, 3 as high amount of collagen in aorta media or in aorta adventitia, and 4 as high amount of collagen in all layers of the aorta. The suprarenal aortic diameter was measured as the adventitial diameter and the definition of an aneurysm was set as a 1.5-fold enlargement of leading edge to leading edge. Measurements of the aortic diameter was performed using Zen 2012 (blue edition, Carl Zeiss Microscopy GmbH, 2011 ver 1.1.2.0, Oberkochen, Germany). To measure aortic outer diameter, the largest dilated part of the aorta was taken for this purpose.
Quantitative real-time PCR Human and mouse aortas were homogenized with trizol and chloroform in a Tissue Lyser using safe-lock tubes with a metal bead. Total RNA from human and mice aortas were isolated with RNeasy mini kit (Qiagen, Hilden, Germany) and reversely transcribed with random primers and Superscript III (Invitrogen, Carlsbad, USA) according to manufacturer0 s instructions. Quantitative real-time polymerase chain reaction (PCR) was performed on ABI 7500 Fast realtime PCR Sequence Detector using gene expression assay primers and 2x TaqMan Fast Universal PCR Mastermix (Applied Biosystems, Foster City, USA). Amplified cDNA (human 0.25 μg; mouse 0.126 μg) was run in duplicates. All probes were obtained from Applied Biosystems and the results were normalized to values of human RPLP0 or mouse Gapdh. Human ADAMTS1, 4, 5, 6, 8, 9, 10, 13, 17 and ADAMTSL-1 and mouse ADAMTS1 were analyzed.
Statistical analysis The statistical analysis was performed with the IMB SPSS Statistics 22. Comparisons of quantitative data were performed using Student0 s t-test and one way analysis of variance (ANOVA). Multiple test correction was performed using Bonferroni post hoc analysis. Pvalues < 0.05 were considered statistically significant where indicates P < 0.05, indicates P < 0.01 and indicates P < 0.001.
Results and discussion mRNA expression of ADAMTS members in AAA mRNA expression of nine of ADAMTS-1, 4, 5, 6, 8, 9, 10, 13, 17 and ADAMTSL-1, were analyzed in aortic wall of aneurysm patients and control aortas using real time RT-PCR (Fig 1). These data show that most of the members are down regulated on mRNA level in aneurysmal aorta compared to control aorta. ADAMTS-1 was one of the family members with the largest reduction reaching a 99% reduction in the expression in the vessel wall under the thrombus
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ADAMTS-1 in abdominal aortic aneurysm
Fig 1. Gene expression of ADAMTS members. Expression in non-aneurysmal control (con) aortas and aneurysmal aorta covered with intraluminal thrombus (TH) and not covered with intraluminal thrombus (NTH). * p
ADAMTS-1 in abdominal aortic aneurysm Emina Vorkapic1, Maggie Folkesson1†, Kerstin Magnell2, Mohammad Bohlooly-Y2, Toste La¨nne3,4, Dick Wågsa¨ter1* 1 Division of Drug Research, Department of Medical and Health Sciences, Linko¨ping University, Linko¨ping, Sweden, 2 Division of Discovery Sciences, Innovative Medicines, AstraZeneca R&D, Mo¨lndal, Sweden, 3 Division of Cardiovascular Medicine, Department of Medical and Health Sciences, Faculty of Health Sciences, Linko¨ping University, Linko¨ping, Sweden, 4 Department of Cardiovascular Surgery, County ¨ stergo¨tland, Linko¨ping, Sweden Council of O † Deceased. * [email protected]
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Abstract Introduction
OPEN ACCESS Citation: Vorkapic E, Folkesson M, Magnell K, Bohlooly-Y M, La¨nne T, Wågsa¨ter D (2017) ADAMTS-1 in abdominal aortic aneurysm. PLoS ONE 12(6): e0178729. https://doi.org/10.1371/ journal.pone.0178729 Editor: Michael Bader, Max Delbruck Centrum fur Molekulare Medizin Berlin Buch, GERMANY Received: February 1, 2017 Accepted: May 17, 2017 Published: June 1, 2017 Copyright: © 2017 Vorkapic et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Swedish Research Council (vr.se, Grant #2016-02626, DW), the Swedish Heart-Lung Foundation (hjartlungfonden.se, Grant#20160779, DW and #20130650, TL) and Åke Wibergs stiftelse (ake.wiberg.se, Grant#M16-0070, DW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Extracellular matrix degradation is a hallmark of abdominal aortic aneurysm (AAA). Among proteases that are capable of degrading extracellular matrix are a disintegrin and metalloproteases with thrombospondin motifs (ADAMTS). Pathogenesis of these proteases in AAA has not been investigated until date.
Methods and results Human aneurysmal and control aortas were collected and analyzed with RT-PCR measuring the ADAMTS-1, 4,5,6,8,9,10,13,17 and ADAMTSL-1. Expression of a majority of the investigated ADAMTS members on mRNA level was decreased in aneurysm compared to control aorta. ADAMTS-1 was one of the members that was reduced most. Protein analysis using immunohistochemistry and western blot for localization and expression of ADAMTS-1 revealed that ADAMTS-1 was present predominantly in areas of SMCs and macrophages in aneurysmal aorta and higher expressed in AAA compared to control aortas. The role of ADAMTS-1 in AAA disease was further examined using ADAMTS-1 transgenic/apoE-/mice with the experimental angiotensin II induced aneurysmal model. Transgenic mice overexpressing ADAMTS-1 showed to be similar to ADAMTS-1 wild type mice pertaining collagen, elastin content and aortic diameter.
Conclusion Several of the ADAMTS members, and especially ADAMTS-1, are down regulated at mRNA level in AAA, due to unknown mechanisms, at the same time ADAMTS-1 protein is induced. The cleavage of its substrates, don’t seem to be crucial for the pathogenesis of AAA but rather more important in the development of thoracic aortic aneurysm and atherosclerosis as shown in previous studies.
PLOS ONE | https://doi.org/10.1371/journal.pone.0178729 June 1, 2017
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ADAMTS-1 in abdominal aortic aneurysm
Competing interests: AstraZeneca R&D provided support in the form of salaries for authors KM and MB-Y, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
Introduction Numerous groups of proteases have been suggested to be involved in the remodelling of the extracellular matrix. The most studied family of matrix-degrading enzymes, which we also contributed to, is probably the matrix metalloproteinases (MMPs) since these proteases are capable to degrade plenty of macromolecules present in the connective tissue matrix [1–3]. The extensive family of matrix metalloproteases beside MMPs includes a disintegrin and metalloproteinase (ADAMs) of which several members have been found in tissue from abdominal aortic aneurysms (AAA) [4, 5]. Closely related to the ADAM family are a disintegrin and metalloproteinase with thrombospondin motifs, (ADAMTS). ADAMTSs are secreted into the extracellular domain and some members of this family, in contrast to other metalloproteases, have capacity to bind to extracellular matrix [6]. ADAMTS similar to other metalloproteases contain a pro-domain that preserves ADAMTSs latency. Not all the substrates for ADAMTSs are known but they usually have various matrix proteins as substrate [7]. ADAMTS-1 is highly expressed in atherosclerotic aorta and promotes atherogenesis by cleaving extracellular matrix proteins such as versican and inducing vascular smooth muscle cell migration [8]. We previously found that ADAMTS-4 and -8 are inflammatory regulated enzymes expressed in LDLR-/-/apoB100/100 mice and in macrophage-rich areas of human atherosclerotic carotid plaques and coronary unstable plaques [9]. AAA occurs when extracellular matrix of the aorta has degenerated, and the aortic diameter enlarges irreversibly with a deadly outcome in the form of rupture. None of the currently known members of ADAMTS family have been studied in relation to AAA. In this study we focused our investigation on the presence and expression of ADAMTS-1. We also included mRNA expression analysis of several other members of our interest such as 4, 5, 6, 8, 9, 10, 13, 17 and ADAMTSL-1 in the aortic wall of control aortas and AAA. The role of ADAMTS-1 in AAA was further investigated using angiotensin II induced AAA in mice. Our data evaluating the expression of ADAMTS members in human aneurysm shows that mRNA expression of several of the members are decreased in AAA samples as compared to control aortas. Mice overexpressing ADAMTS-1 in an angiotensin II induced AAA develop aneurysm to the same extent as wild type mice and therefore ADAMTS-1 probably plays minor role in AAA progression.
Materials and methods Human samples AAA patients (n = 16, all men with mean age of 66, range 56–74 with aortic diameter of 65 mm, range 47–75) undergoing surgical repair were included in this study and biopsies from thrombus-free (NTH, n = 12) and thrombus-covered (TH, n = 16) aneurysm wall were taken since previous studies have revealed major differences in inflammatory response between aneurysm walls covered and not covered by an intraluminal thrombus [2]. Samples included were sufficient to detect a difference of two-fold with 95% confidence interval and at >80% power. Aortic walls were assayed as whole aorta and part of them were dissected to adventitia and media in aneurysmal aorta. Aorta from organ donors (n = 13, all men with mean age of 49, range 32–61) without clinical or macroscopic signs of aortic atherosclerosis or aneurysm were used as non-aneurysmal external controls. Biopsies were immediately fixed in 4% formaldehyde for histology analyses, snap frozen in liquid nitrogen for protein analysis or placed in RNAlater (Ambion, Austin, TX, USA) overnight in 40 C and then stored in -800 C for RNA isolation.
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ADAMTS-1 in abdominal aortic aneurysm
All AAA participants gave written informed consent to the study, which was approved by the regional ethical review board in Linko¨ping, Sweden. The use of organ donors was approved by the regional ethical review board in Lund, Sweden.
AngII-induced AAA in mice AngII-induced aneurysm is an inflammation-driven model that is frequently used to experimentally induce AAA [10–12]. Aneurysm was induced in wild type male hypercholesterolemic apoE-/- mice (Taconic, Bomholt, Denmark) mice (n = 12) and apoE-/-/ADAMTS-1 transgenic mice (n = 14), established and obtained from Astrazeneca, Gothenburg, Sweden as previously described [8]. Animals included were sufficient to detect a 20% difference with 95% confidence interval at >80% power. Animals were housed in groups in pathogen free (SPF) facility in IVC enriched cages and maintained on a 12:12 hour light/dark cycle. At eight weeks of age, AAA was induced, in randomly chosen mice, by chronic infusion of 1000 ng/kg/min AngII (Cat.no.9525, Sigma Aldrich, St. Louis, USA) via mini-osmotic pumps (Model 1004, Alzet, CA, USA) as described previously [11]. Pumps were placed under anesthesia using isoflurane. Post-operative pain was treated with buprenorphine at a dose of 0.1 mg/ kg body weight, injected subcutaneously twice per day for 3 days. A group of apoE-/- mice were infused with 0.9% NaCl and were used as control mice. Standard Chow diet and water was allowed ad libidum throughout the whole study and mice were monitored daily for signs of discomfort. After 28 days, mice were sacrificed. The aorta was removed and fixated in RNAlater for 24 hours thereafter frozen in -70˚C for gene expression analysis or snap frozen for protein analysis. Part of aorta was fixed in formaldehyde and embedded in paraffin for histology and analysis of aneurysm formation. The study was approved by the local ethical committee in Linko¨ping, Sweden (47–13).
Immunohistochemistry Paraffin-embedded human abdominal aortas from four patients were sectioned (5 μm) and rehydrated in several changes of ethanol and Tissue-Clear1 (Sakura Finetek, Leiden, The Netherlands). Endogenous peroxidase activity was quenched by treatment with 3% hydrogen peroxide for 5 min followed by incubation in 5% blocking rabbit or goat serum albumin solution. Sections were then incubated with human primary antibodies against ADAMTS-1 (2 μg/ ml, Cat. No. AF5867, R&D Systems, Inc, Minneapolis, USA), CD68 (0.1 μg/ml, Cat. No. NCL-CD68-KP1, Leica Microsystems, Newcastle, UK) and α-actin (0.6 mg/ml, cat. No. A5228, clone 1A4, Sigma-Aldrich) at 4˚C overnight followed by secondary biotinylated rabbit anti-sheep IgG or goat anti-mouse IgG antibody (Dako, Stockholm, Sweden). Avidin-biotin peroxidase complexes (Dako) were added followed by visualization with 3,30 -diaminobenzidine tetrahydrochloride (Dako). All sections were counterstained with Mayer’s hematoxylin (Histolab Products, Go¨teborg, Sweden).
Western blot Frozen aortas from AAA patients and non-aneurysmal control donors were homogenised in ice-cold RIPA lysis buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% Na-deoxy-cholate, 1% Triton-100 and 0.1% SDS, 100 μg/ml phenylmethylsulphonyl fluoride (PMSF) and protease inhibitor cocktail (Roche Diagnostics Scandinavia, Stockholm, Sweden). Protein quantification of tissue extracts (30 μg of protein) were separated under reducing conditions using 4–12% gradient SDS-PAGE and transferred onto a polyvinylidine difluoride membrane (Amersham Life Science, Amersham, UK). Membranes were blocked in 5% non-fat dry milk and incubation with primary rabbit ADAMTS-1 antibody (1 μg/ml, Cat.
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ADAMTS-1 in abdominal aortic aneurysm
No. AF5867, R&D Systems) overnight at 4˚C followed by incubation by a horseradish peroxidase-conjugated secondary anti-sheep antibody (1:5000, Cat. No. sc-2770, Santa Cruz Biotechnology, Heidelberg, Germany). Immunoreactive proteins were visualized by chemiluminescence ECL Prime Western blotting detection system (Amersham). Loading was controlled using coomassie blue staining of membranes.
Elastin and collagen staining Paraffin-embedded sections (5 μm) from mice aorta were rehydrated in several changes of ethanol and Tissue-Clear1 (Sakura Finetek). Sections were then stained for collagen and elastin as previously described [13]. Sections were studied under light microscopy ZEN 2012 (Zeiss, Jena, Germany). Quantification of collagen and elastin were performed blinded. A scoring system from 1 to 4 was used with 1 defined as no degradation of elastin, 2 as low degradation, 3 as intermediate degradation, and 4 as high degradation. For collagen quantification, 1 defined as low amount of collagen in all layers of the aorta, 2 as low amount of collagen in aorta media or in aorta adventitia, 3 as high amount of collagen in aorta media or in aorta adventitia, and 4 as high amount of collagen in all layers of the aorta. The suprarenal aortic diameter was measured as the adventitial diameter and the definition of an aneurysm was set as a 1.5-fold enlargement of leading edge to leading edge. Measurements of the aortic diameter was performed using Zen 2012 (blue edition, Carl Zeiss Microscopy GmbH, 2011 ver 1.1.2.0, Oberkochen, Germany). To measure aortic outer diameter, the largest dilated part of the aorta was taken for this purpose.
Quantitative real-time PCR Human and mouse aortas were homogenized with trizol and chloroform in a Tissue Lyser using safe-lock tubes with a metal bead. Total RNA from human and mice aortas were isolated with RNeasy mini kit (Qiagen, Hilden, Germany) and reversely transcribed with random primers and Superscript III (Invitrogen, Carlsbad, USA) according to manufacturer0 s instructions. Quantitative real-time polymerase chain reaction (PCR) was performed on ABI 7500 Fast realtime PCR Sequence Detector using gene expression assay primers and 2x TaqMan Fast Universal PCR Mastermix (Applied Biosystems, Foster City, USA). Amplified cDNA (human 0.25 μg; mouse 0.126 μg) was run in duplicates. All probes were obtained from Applied Biosystems and the results were normalized to values of human RPLP0 or mouse Gapdh. Human ADAMTS1, 4, 5, 6, 8, 9, 10, 13, 17 and ADAMTSL-1 and mouse ADAMTS1 were analyzed.
Statistical analysis The statistical analysis was performed with the IMB SPSS Statistics 22. Comparisons of quantitative data were performed using Student0 s t-test and one way analysis of variance (ANOVA). Multiple test correction was performed using Bonferroni post hoc analysis. Pvalues < 0.05 were considered statistically significant where indicates P < 0.05, indicates P < 0.01 and indicates P < 0.001.
Results and discussion mRNA expression of ADAMTS members in AAA mRNA expression of nine of ADAMTS-1, 4, 5, 6, 8, 9, 10, 13, 17 and ADAMTSL-1, were analyzed in aortic wall of aneurysm patients and control aortas using real time RT-PCR (Fig 1). These data show that most of the members are down regulated on mRNA level in aneurysmal aorta compared to control aorta. ADAMTS-1 was one of the family members with the largest reduction reaching a 99% reduction in the expression in the vessel wall under the thrombus
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ADAMTS-1 in abdominal aortic aneurysm
Fig 1. Gene expression of ADAMTS members. Expression in non-aneurysmal control (con) aortas and aneurysmal aorta covered with intraluminal thrombus (TH) and not covered with intraluminal thrombus (NTH). * p
![[The abdominal aortic aneurysm].](/assets/img/hold.png)
